Insulin production by the B-cell consists of a series of post- translational events: introduction of preproinsulin into the lumen of the RER; prepro- to proinsulin conversion; transport of proinsulin to the Golgi complex and packaging into immature, clathrin coated granules; proinsulin to insulin conversion. granule acidification and clathrin uncoating; granule release, storage or degradation. The ultimate aim of this Project is to characterize each step in molecular detail. Only then will it be possible to define the lesions responsible for defective insulin production in diabetes. The experiments depend upon native islets (rat or calf) or rat insulinoma cells. The methods combine analytical biochemistry, cell biology, morphology and recombinant DNA techniques. The specific aims and experimental approaches are: 1. Introduction of preproinsulin into the RER: The signal (leader) sequence carries structural information responsible for penetration through the RER membrane. This region of the insulin gene will be mutated (site directed mutagenesis of rat insulin II gene) and the mutated gene transfected into AtT20 cells (pituitary cell line). Production of insulin and its precursors will be compared with AtT20 cells transfected with the native gene. Relative rates of synthesis, conversion and intracellular transport/targeting/packaging will be examined. 2. Targeting/packaging of proinsulin into granules, and subsequent conversion to insulin: A similar approach will be used to study the tertiary and quarternary structural information on the proinsulin molecule responsible for recognition in the Golgi complex and by the converting enzyme(s). 3. Granule maturation (clathrin uncoating), intragranular acidification and proinsulin conversion: Isolated, intact granules will be used. The mechanism responsible for removal of clathrin from immature granules will be studied. The experiments will also address the issue of whether uncoating, acidification and conversion are merely contemporaneous or actually interdependent events. 4. The preperential release of newly synthesized insulin: The mechanism underlying preferential release of new insulin will be studied using native rat islets. The hypothesis that intimate association of newly formed granules with the cytoskeleton is responsible will be evaluated by examining the effect of agents known to disturb microtubule polymerization. 5. Intracellular degradation of granule contents: After fusion of granules with lysosomes (crinophagy) insulin is predicted to be degraded less rapidly than C-peptide/proinsulin (since it is stabilized in the crystal form). The rates of insulin and C-peptide degradation will be measured in rat islet B-cells. How granules and lysosomes come in contact and fuse is unknown. The hypothesis that the cytoskeleton serves as the framework for crinophagy will be tested experimentally.